Inhibition of adenylyl cyclase by neuronal P2Y receptors.

1 P2Y receptors inhibiting adenylyl cyclase have been found in blood platelets, glioma cells, and endothelial cells. In platelets and glioma cells, these receptors were identified as P2Y(12). Here, we have used PC12 cells to search for adenylyl cyclase inhibiting P2Y receptors in a neuronal cellular environment. 2 ADP and ATP (0.1 - 100 mum) left basal cyclic AMP accumulation unaltered, but reduced cyclic AMP synthesis stimulated by activation of endogenous A(2A) or recombinant beta(2) receptors. Forskolin-dependent cyclic AMP production was reduced by less than or equal to1 pm and enhanced by 10 - 100 mum ADP; this latter effect was turned into an inhibition when A(2A) receptors were blocked. 3 The nucleotide inhibition of cyclic AMP synthesis was not altered when P2X receptors were blocked. but abolished by pertussis toxin. 4 The rank order of agonist potencies for the reduction of cyclic AMP was (IC50 values): 2-methylthio-ADP (0.12 nM) = 2-methylthio-ATP (0.13 nM) > ADPbetaS (71 nM) > ATP (164 nM) = ADP (244 nM). The inhibition by ADP was not antagonized by suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid, or adenosine-3'-phosphate-5'-phosphate, but attenuated by reactive blue 2, ATPalphaS, and 2-methylthio-AMP. 5 RT-PCR demonstrated the expression of P2Y(2), P2Y(4), P2Y(6), and P2Y(12), but not P2Y(1), receptors in PC12 cells. In Northern blots, only P2Y(2) and P2Y(12) were detectable. Differentiation with NGF did not alter these hybridization signals and left the nucleotide inhibition of adenylyl cyclase unchanged. 6 We conclude that P2Y(12) receptors are expressed in neuronal cells and inhibit adenylyl cyclase activity.